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Targeted fibrillar nanocarbon RNAi treatment of acutekidney injurySimone Alidori,1 Nima Akhavein,1 Daniel L. J. Thorek,2 Katja Behling,1 Yevgeniy Romin,3

Dawn Queen,1 Bradley J. Beattie,4 Katia Manova-Todorova,3 Magnus Bergkvist,5

David A. Scheinberg,6,7,8 Michael R. McDevitt1,7*

RNA interference has tremendous yet unrealized potential to treat a wide range of illnesses. Innovative solutions areneeded to protect and selectively deliver small interfering RNA (siRNA) cargo to and within a target cell to fullyexploit siRNA as a therapeutic tool in vivo. Herein, we describe ammonium-functionalized carbon nanotube(fCNT)–mediated transport of siRNA selectively and with high efficiency to renal proximal tubule cells in animalmodels of acute kidney injury (AKI). fCNT enhanced siRNA delivery to tubule cells compared to siRNA alone andeffectively knocked down the expression of several target genes, including Trp53,Mep1b, Ctr1, and EGFP. A clinicallyrelevant cisplatin-induced murine model of AKI was used to evaluate the therapeutic potential of fCNT-targetedsiRNA to effectively halt the pathogenesis of renal injury. Prophylactic treatment with a combination of fCNT/siMep1b and fCNT/siTrp53 significantly improved progression-free survival compared to controls via a mechanismthat required concurrent reduction of meprin-1b and p53 expression. The fCNT/siRNA was well tolerated, and notoxicological consequences were observed in murine models. Toward clinical application of this platform, fCNTswere evaluated for the first time in nonhuman primates. The rapid and kidney-specific pharmacokinetic profile offCNT in primates was comparable to what was observed in mice and suggests that this approach is amenable foruse in humans. The nanocarbon-mediated delivery of siRNA provides a therapeutic means for the prevention ofAKI to safely overcome the persistent barrier of nephrotoxicity during medical intervention.

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INTRODUCTION

RNA interference (RNAi) is acknowledged as an important techno-logical advance promising new therapeutic strategies through generegulation. One mechanism of interference involves sequence-specifictargeting of messenger RNA (mRNA) with complementary small in-terfering RNA (siRNA) resulting in mRNA degradation. Accordingly,siRNA-based therapeutics can transiently mute genes that regulate dis-ease or injury. However, siRNA has yet to overcome major obstaclesin vivo primarily related to tissue- and cell-specific delivery of siRNA,untoward off-target effects, and poor serum stability (1, 2). Nano-molecular delivery platforms are being investigated as a means toovercome the obstacles to use siRNA in vivo (2–5). An ideal platformis expected to be biocompatible and nonimmunogenic, possess a ca-pacity for transport of RNA cargoes to the target cell, and afford pro-tection from ribonucleases. Nanoscale molecular transporters andsynthetic modification of the RNA backbone may remedy some of theseproblems, and several lipid and polymer nanoparticle formulationshave already entered clinical trial (2–5).

Carbon nanotubes have been investigated as siRNA delivery plat-forms (6, 7). Ammonium-functionalized single-walled carbon nano-tubes (fCNTs) are a unique class of fibrillar macromolecules that candeliver drugs, proteins, and radioisotopes (8). Paradoxically, owing to

1Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065,USA. 2Division of Nuclear Medicine and Molecular Imaging, Department of Radiology andRadiological Science, Johns Hopkins University School of Medicine, Baltimore, MD 21205,USA. 3Molecular Cytology Core Facility, Memorial Sloan Kettering Cancer Center, NewYork, NY 10065, USA. 4Department of Medical Physics, Memorial Sloan Kettering CancerCenter, New York, NY 10065, USA. 5College of Nanoscale Science and Engineering, Uni-versity at Albany, Albany, NY 12203, USA. 6Department of Molecular Pharmacology, Me-morial Sloan Kettering Cancer Center, New York, NY 10065, USA. 7Department ofMedicine, Weill Cornell Medical College, New York, NY 10065, USA. 8Department of Phar-macology, Weill Cornell Medical College, New York, NY 10065, USA.*Corresponding author. E-mail: m-mcdevitt@ski.mskcc.org

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their large aspect ratio, fCNTs have a very favorable renal glomerularfiltration and elimination profile (9–12), unlike most of the globularparticles that accumulate in the liver and/or do not clear. A fractionof filtered fCNT is reabsorbed at the kidney’s proximal tubular cell(PTC) brush border and endocytosed (10). This provides the oppor-tunity for fCNT to transport noncovalently bound siRNA (Fig. 1A) toand within the critical PTC physiological compartment (13) and thustreat kidney-related pathologies.

Acute kidney injury (AKI) is recognized as an unavoidable sideeffect of numerous medical treatments. These include nephrotoxic dam-age sustained by antibiotics, antivirals, and chemotherapy as well assurgical procedures, which deprive the kidney of oxygen (14, 15). Injuryto this organ is exacerbated in the elderly, which make up the bulk ofthe cancer population. The result is protracted and expensive hospitalcare, and half of the elderly population with AKI will succumb. Thissevere morbidity limits the therapeutic window because chemotherapeuticdosages must be titrated down, resulting in a reduced antineoplasticeffect. The pathogenesis of AKI is a complex biological process (15),and the loss of proximal tubule cell polarity (16, 17) and apoptosis(18, 19) are critical early events. Currently, treatment of AKI is largelysupportive after damage, and despite the large number of patients atrisk, pharmacological therapies remain unavailable (20).

Meprin-1b and p53 are key proteins in the depolarization andapoptotic processes of kidney injury, respectively. p53 promotes cellcycle arrest or apoptosis in response to cellular stress, whereas meprinsare metalloproteinases localized to the brush border membrane ofpolarized epithelial cells, where they are able to hydrolyze peptidesand extracellular proteins (21). A redistribution of meprin to the cyto-sol in response to an insult is associated with renal injury (22). In studieswhere meprin activity was inhibited, there was protection against AKIinduced by ischemia-reperfusion (I/R) injury, cisplatin nephrotoxicity,and sepsis (23–25). Meprin-deficient mice were markedly resistant to

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kidney damage from I/R. A chemically modified siRNA targeted top53 was previously investigated to prevent kidney injury (19) and wasevaluated clinically but did not meet the primary endpoint in a phase 2clinical trial (4). Knocking out p53 in mice has also been reported toimprove survival in response to nephrotoxic insults (26). However, nostudy has looked at this combination RNAi to prevent AKI.

Here, we demonstrate specific delivery of Trp53- andMep1b-targetedsiRNA to proximal tubule cells using an fCNT platform to prophylac-tically mitigate AKI in animal models. The fCNT-facilitated siRNAdelivery prevented renal injury after a nephrotoxic insult that sub-sequently reduced fibrosis and immune cell infiltration and resultedin progression-free survival. Our data also provide strong evidencefor the role of meprin in AKI. Toward clinical application of fCNT/siRNA for targeted prophylactic AKI treatment, we also evaluatedfCNT biodistribution in nonhuman primates. A clinical strategyusing the fibrillar nanocarbon platform could enable targeted siRNAprotection of the kidney safely and effectively and prevent AKI in thoseon chronic pharmacological regimens, especially the elderly andcancer patients.

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RESULTS

Characterization of the fCNT/siRNA complexThe fCNTs were prepared and characterized as previously reportedvia covalent cycloaddition of azomethine ylides with single-walledCNT (SWCNT) (9–13). fCNT had an amine content of 0.3 mmol/g(fig. S1A) and chemical purity >99%. Dicer-validated RNA sequences(27) were designed to silence enhanced green fluorescent protein (EGFP),murine copper transport protein 1 (Ctr1), meprin-1b (Mep1b), and p53(Trp53); a nonspecific scrambled sequence (Scram) was used as a con-trol. The noncovalent binding affinity between fCNT and siRNA was~5 nM (Fig. 1B), and up to four siRNAs could be loaded per fCNT(Fig. 1C) under physiological conditions (13). Transmission electronmicroscope (TEM) imaging of solid fCNT and fCNT/siEGFP (1:1 molarratio) showed an average length of 300 nm (Fig. 1D). Both fCNTsamples were water-soluble up to 10 g/liter, could be resolved chromato-graphically, and were rapidly renally filtered in mice, as has been shownpreviously (9–11). The molecular lengths of fCNT and fCNT/siEGFP(at 1:1 molar ratio) were comparable, with mean diameters (±SD) of 356.2± 14.2 and 332.7 ± 10.6 nm, respectively (fig. S1B). High-performanceliquid chromatography (HPLC) showed a single peak (330 nm) (fig. S1C).

Kinetics of fCNT-mediated siRNA transport in vitroCellular internalization of fCNT/siEGFP-Cy3 (1:1) was investigated withEGFP-expressing HeLa cells (EGFP+ HeLa) using time-lapse confocalmicroscopy. In accordance with the loading/off-loading mechanismfor fCNT/siRNA [when concentration is greater than the dissociationconstant (Kd), then the two species remain bound together with nano-molar affinity; at concentrations below theKd (such as when the complexis internalized inside the cell and the local concentration is lower thanthe extracellular milieu), the siRNA will dissociate from the nanotube(13)], fCNT/siEGFP-Cy3 did not fluoresce due to cyanine emissionquenching by fCNT. siRNA-Cy3 emission or siRNA-[111In] signal wasdetected at 2 hours and progressively increased over the 5-hour timecourse experiment (fig. S2A), as the fCNT complex was internalizedand the siRNA off-loaded intracellularly. siEGFP alone exhibited neg-ligible internalization. The internalization radioassay indicated that 104

Fig. 1. Assembly of the CNT siRNA construct. (A) An illustration of thenoncovalent bonding interactions between an fCNT and a siRNA to yield

the molecular RNAi construct (n.b. not to scale). (B) Plot of the fluorescencequenching titration of siEGFP-Cy3 with fCNT and fitted binding isotherm(dashed line). (C) Relative fluorescence intensity as a function of siEGFP-Cy3/fCNT molar ratio and graphical interpolation of the curve (dashed lines)to yield the siEGFP/fCNT loading stoichiometry. (D) Representative TEMimages of solid-state fCNT and fCNT/siEGFP (1:1 complex). Scale bars,500 mm.

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molecules of siRNA were delivered by fCNT per cell, whereas siRNAalone demonstrated minimal uptake (fig. S2B).

fCNT-mediated RNAi of EGFP in vitroGene and protein knockdown was evaluated in EGFP+ HeLa cells.Time-lapse confocal microscopy images were collected over 60 hours,and region of interest (ROI) analyses showed that fCNT/siEGFP and aLipofectamine/siEGFP (Lf/siEGFP)–positive control produced a signif-icant decrease in fluorescence; control siEGFP alone was visibly lesseffective (Fig. 2A). The fCNT-mediated siEGFP reduced fluorescenceexpression by 70% at 24 hours and by 92% at 60 hours, whereas con-trol siEGFP alone reached maximal inhibition of about 40% by 60 hours(Fig. 2A). Several cell division cycles were imaged and confirmed bothcell viability and biocompatibility of the fCNT transfection reagent(fig. S3A). Confirmation of fCNT-mediated interference was acquiredat 1, 2, and 3 days using flow cytometry (Fig. 2B), Western blot (Fig.2C), or reverse transcription polymerase chain reaction (RT-PCR)(Fig. 2D); each demonstrated a decrease in expression compared tocontrol groups. Notably, fCNT/siEGFP reduced EGFP expression sig-nificantly more than Lf/siEGFP. Kinetic analysis indicated that themaximum RNAi occurred at 48 hours (fig. S3B). Cytotoxicity wasevaluated as a function of increasing dose of fCNT (or controls); after

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72 hours of constant exposure, there was no significant difference com-pared to the Lipofectamine group up to a concentration of 100 mg/liter(fig. S4).

The pharmacokinetic profile of fibrillar nanocarbon andsiRNA cargoSpecific renal targeting of fCNT/siRNA was substantiated by evaluat-ing the pharmacokinetic (PK) fate in mice. As the main tissue tar-geted, kidneys accumulated about 22% of the injected dose of fCNT/siEGFP-[111In]DOTA per gram within 1 hour (Fig. 3A and fig. S5),indicating that the siRNA remained bound to the fCNT in vivo until itreached the kidney. fCNT-mediated delivery resulted in a 10-fold in-crease of siRNA kidney accumulation compared to control, and the frac-tion of dose that was not delivered to the kidney was rapidly (<1 hour)eliminated (fig. S5B). fCNT also protected siRNA cargo from serumdegradation (fig. S6).

Renal cortex uptake of fCNT was confirmed using confocal mi-croscopy (Fig. 3B). Organelle trafficking was investigated, and repre-sentative images of the early endosome, Golgi, and lysosomes in thecortex all costained for fCNT; early endosome signal was evidenced at5 min, whereas Golgi and lysosome staining was more pronounced at1 hour (Fig. 3C). These data, along with our previous reports (10, 28),

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support a clathrin-mediated endocyticuptake mechanism by the PTC (29, 30).

The biodistribution of tracer-labeled[86Y]fCNT was determined in a large ani-mal model (naïve nonhuman primates)using positron emission tomography–computed tomography (PET/CT). fCNTexhibited similar blood clearance, tissue bio-distribution, and renal elimination in a 5-kgcynomolgus monkey (Macaca fascicularis).Intravenous [86Y]fCNT (1 mg/kg) had ablood half-life of 7 min. Majority of thedose was rapidly eliminated in the urine,with a fraction accumulated in the kidneys(standardized uptake value was 16) (Fig. 3D).

Mitigating AKI with kidney-targeted fCNT-mediated RNAiA therapeutically effective dose of fCNT/siRNA inmice was determined to be 1.6-mgfCNT + 0.087-mg siRNA per kilogram perday for 3 to 5 days (Fig. 1B) (13). Thesedoses of fCNT/siRNA were sufficient toachieve knockdown of target mRNAs andwere well tolerated by the host. Our regi-men achieved prophylaxis with a cumu-lative dose of 0.4 mg siRNA/kg. We firstdemonstrated renal tubule–specific geneknockdown using fCNT and siRNA tar-geting EGFP (31). fCNT/siEGFP yielded asignificant 75% decrease in renal corticalfluorescence versus controls receivingsiEGFP alone, a scrambled siRNA, or ve-hicle control (Fig. 4, A to C). Western blotconfirmed fCNT-mediated knockdown(Fig. 4D).

Fig. 2. In vitro functional study of fCNT-mediated siEGFP delivery to EGFP+ HeLa cells. (A) Repre-sentative time-lapse confocal microscopy images at 1, 24, and 60 hours. Fluorescence quantification from

three ROIs of the cells. Scale bars, 50 mm. (B) Flow cytometry histogram overlay. (C and D) Western blot (C)and RT-PCR analysis (D) of EGFP expression by cells isolated at day 3 after transfection. Data in (A) and (D)are means ± SEM (n = 3). P values were determined by unpaired t test. h, hours; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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EGFP knockdown mediated by fCNT/siRNA provided the in vivoproof of principle. We therefore attempted to prophylactically amelio-rate AKI by down-regulating Ctr1, a transmembrane protein respon-sible for the cellular uptake of copper that has been implicated as thekey mediator of cisplatin uptake into the renal tubule (32). The quan-tification of renal accumulation of copper-64 served as a functionalreadout of Ctr1 expression. Animals treated with fCNT/siCtr1 showeda significant decrease in renal copper uptake at 1 hour after injectioncompared to the untreated group and the siCtr1 alone (Fig. 4E). siCtr1cargo administered without fCNT transport was unable to decreasecopper uptake.

Safely targeting two genes involved in AKI pathogenesisMeprin-1b and p53 proteins play key roles in the depolarization andapoptotic processes of kidney injury, respectively. We aimed to targetthe genes encoding these proteins, Mep1b and Trp53, to establish fea-sibility of fCNT-mediated RNAi in AKI and to evaluate biocompatibil-ity of the siRNA delivery system. Each animal received the fCNT/siRNAconstructs or the siRNA alone at a dose of 0.087-mg siRNA ± 1.6-mgfCNT per kilogram per day for three consecutive days. Immunohisto-chemistry (IHC) revealed that the fCNT-mediated RNAi reduced theexpression of both target proteins in the cortex (Fig. 5A). QuantitativeROI analysis of the IHC images revealed a significant decrease in p53expression in the fCNT/siTrp53 group versus siTrp53 alone and vehicle.Similarly, meprin-1b expression was significantly reduced by fCNT/siMep1b, but not when receiving siMep1b alone or vehicle (Fig. 5B).

Neither the nanocarbon vehicles nor the siRNA adversely affectedrenal health or tissue morphology. Renal function was assessed using ametabolic panel of blood urea nitrogen, serum creatinine, and phos-phorus as biomarkers. No statistical changes were observed for any ofthe biomarkers, indicating that prophylactic fCNT and/or siRNA com-ponents were biocompatible (table S1). Tissue morphology was ex-amined and scored (fig. S7) with no structural abnormalities.

Combination siTrp53 and siMep1bminimizedmRNA and proteinexpression in kidneys challenged with a nephrotoxic insultMice were prophylactically treated for five consecutive days with acombination dose of fCNT/siTrp53/siMep1b or scrambled control

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Fig. 3. PK profile of fibrillar nanocarbon and siRNA in mice, PTC or-ganelle trafficking, and PET/CT imaging of [86Y]fCNT in a nonhuman

primate. (A) Tissue biodistribution of fCNT/siEGFP-[111In]DOTA and siEGFP-[111In]DOTA at 1 hour after injection. The y axis shows the percent of theinjected dose per gram (%ID/g) of tissue. Data are means ± SEM (n = 5). P valuedetermined by unpaired t test. (B) Representative immunofluorescence (IF)images of 5-mm kidney sections stained for Alexa Fluor 488 to mark fCNT(green) and DAPI (4′,6-diamidino-2-phenylindole) to mark nuclei (blue) ofmice administered with fCNT-AF488 and sacrificed after 1, 3, 7, and 30 days.Scale bars, 100 mm. The panel also includes the quantification of the AF488signal. Data are means ± SEM (n = 6 ROIs). (C) Confocal microscopy imagesof fCNT colocalization with different organelle staining (red) at 5 min, 20 min,60 min, and 24 hours from mouse tissue. Images of the early endosome,Golgi apparatus, and lysosomes were obtained with EEA-1, GM130, andLAMP-1 co-staining, respectively. Scale bars, 5 mm. (D) Fused PET/CT imageof a representative 5-kg cynomolgus monkey (M. fascicularis) that receivedan intravenous bolus of [86Y]fCNT (1 mg/kg) and quantitative analysis of thestandard uptake value (SUV) in the kidney and bladder. The PET data arerepresented across the frames using the identical semiquantitative blue(low) to red (high) color scale. (E) PET/CT image of transverse kidney sec-tion taken where the white dotted lines are in (D).

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(siScram) and then presented with a single nephrotoxic dose of cis-platin to induce AKI (10 mg/kg) on the third day. Mice were sacrificedon day 5, and IHC staining of paraformaldehyde (PFA)–fixed kidneytissues showed low p53 and meprin-1b signals in the naïve controlsand elevated protein expression in the fCNT/siScram group. The com-bination drug maintained both p53 and meprin-1b at baseline ex-pression (Fig. 6A). Protein quantification in kidney cortexes via panenzyme-linked immunosorbent assay (ELISA) (Fig. 6B) and mRNAexpression (Fig. 6C and fig. S8) confirmed the IHC data demonstrat-ing simultaneous Trp53 and Mep1b knockdown. Fluorescence in situhybridization (FISH) microscopy imaging permitted more accurateidentification of mRNA in the renal cortex, and representative imagesof Trp53,Mep1b, and dapBmRNA in mouse cortical PTCs are shownin fig. S8A.

Simultaneously targeting Trp53 and Mep1b reduced renalinjury, fibrosis, and immune infiltrationFibrillar nanocarbon-mediated RNAi treatment successfully minimizedrenal injury from a nephrotoxic cisplatin dose and improved progression-free survival in mice by targetingMep1b and Trp53 in the PTC. RNAi

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treatment or control was administereddaily for five consecutive days, com-mencing on day −2, as outlined in theexperimental timeline in Fig. 7A. A sin-gle dose of cisplatin (10 mg/kg) was ad-ministered on day 0. The cisplatin dose wasselected on the basis of a dose-responsestudy (fig. S9), whereas the nanotube-RNAi drug dosage was established on thebasis of the binding affinity (Fig. 1B) anda dose escalation study (fig. S10).Animals were monitored for acute injurydays 1 through 11 after cisplatin and thenlongitudinally over 6 months for chronicor adverse effects (tables S2 and S3). Kid-neys obtained from randomly selectedmice were histologically examined at 11and 180 days for fibrosis and immune cellinfiltration.

The fCNT/siTrp53/siMep1b combina-tion prolonged injury-free survival signif-icantly compared to all controls (Fig. 7B).Eighty-eight percent of the animals treatedwith the combination fCNT/siRNA sur-vived injury-free for the full 11 days aftercisplatin, whereas <40% of those treatedwith fCNT/siMep1b, siMep1b, and fCNT/siTrp53 survived that long. Median timesto injury and the comprehensive resultsof statistical analyses are reported in tableS4. These data describe a prophylactictherapeutic intervention for acute renalinjury that depends on fCNT-mediateddelivery of siRNA targeting two earlypathologic events. A forest plot of thehazard ratios strongly favored the fCNT/siTrp53/siMep1b combination drug inminimizing renal injury (Fig. 7C). There

was no therapeutic advantage in the separate use of fCNT/siMep1bor fCNT/siTrp53, and the siRNA vectors alone were ineffective pre-sumably because of degradation and/or low delivery efficiency. ThefCNT/siCtr1 therapy was also ineffective in minimizing injury andimproving survival (Fig. 7B).

Histological analysis of kidneys from fCNT/siTrp53/siMep1b- andfCNT/siScram-treated mice was performed at 11 and 180 days afternephrotoxic injury. Hematoxylin and eosin (H&E)–stained tissuefrom mice that received the combination drug showed tissue mor-phology consistent with healthy control mouse tissue (Fig. 7D). Kid-ney fibrosis was not observed at 11 days in any animal, but theinterstitial fibrotic level was significantly higher for the fCNT/siScramgroup at 180 days (Fig. 7E). Lymphocyte and macrophage infiltrationoccurs in both the early and later phases of cisplatin-induced AKI (33, 34);therefore, immune infiltration was assessed using IF staining for CD45+

leukocytes, CD3+ T lymphocytes, and Iba1+ macrophages. fCNT/siTrp53/siMep1b treatment significantly reduced T cell, lymphocyte,and macrophage infiltration as early as 11 days after cisplatin injury, withthe cell populations remaining at lower densities than scrambled controlsat 180 days (Fig. 7, F to H).

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Fig. 4. In vivo proof of concept of nanocarbon-mediated RNAi. (A) Representative images of kidney cryo-sections from an EGFP transgenic mouse model treated forthree consecutive days with the indicated siRNA or vehiclecontrol, sacrificed and imaged at day 4. Scale bars, 500 mm.

(B) Quantification of EGFP-positive cells and number of nuclei in individual tubule cells. (C) Ratio of EGFP-positive cells to total cells in the PTC as a function of treatments. Data in (B) and (C) are means ± SEM(n = 300 cells per group). (D) Western blots of the kidney cortex tissues confirming fCNT/siEGFPknockdown of protein expression. (E) In vivo proof of principle for fCNT-mediated RNAi of transporterfunction. Copper transporter loss of function in Balb/c mice treated with fCNT/siCtr1, siCtr1 alone, orvehicle control evaluated through renal 64CuCl2 uptake at1 hour after administration and expressed as %ID/g. Data are individual kidneys with means ± SEM (n = 9). P values in (C) and (E) were determined byunpaired t test.

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DISCUSSION

Here, we deployed CNTs to deliver bioactive siRNA to renal PTC as apharmacological intervention to prevent nephrotoxic injury. Despitegreat potential, use of therapeutic RNAi is hindered by ineffectivedelivery strategies, degradation, and off-target effects. Currently, there

Fig. 5. Reduction in renal cortex expression of p53 andmeprin-1b afterfCNT-mediated RNAi.Mice (n = 3) were treated daily for three consecutive

days with siRNA targeting Trp53 orMep1b. Animals were sacrificed on day 4,and tissues were stained for protein expression. (A) Representative IHCimages of p53 and meprin-1b in cortical kidney sections, including immu-noglobulin G (IgG) negative control. Scale bars, 20 mm. (B) Quantitative ROIanalysis of p53 and meprin-1b IHC images. Data are means ± SEM (n = 15ROIs per section). P values were determined by unpaired t test.

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Fig. 6. Combination fCNT/siRNAminimizesmRNA and protein expressionin vivo during cisplatin-induced renal injury. Mice were treated for five con-

secutive days with PBS (phosphate-buffered saline), fCNT/siScram, or fCNT/siTrp53/siMep1b. On day 3, fCNT-treated mice also received cisplatin (10 mg/kg),whereas controls received saline. All animals were sacrificed on day 6. (A) Rep-resentative p53 andmeprin-1b IHC images of cortical kidney sections. Scale bars,20 mm. (B) Quantitative analysis of the pan ELISA assay for p53 and meprin-1b.Data aremeans± SEM (n=3 for thenaïve groups and4 for the other groups).(C) Quantitative analysis of the FISH for Trp53 and Mep1b mRNA. Data aremeans ± SEM (n = 9). P values were determined by unpaired t test.

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Fig.7. Kidney-targetedfCNT/siRNA improves

survival andminimizesfibrosis and immunecell infiltration aftera drug-induced injury.(A) Timeline of theprogression-free survi-val experiment detailingthe RNAi prophylaxistreatments, cisplatinadministration, serumsampling for bloodchemistry analysis,weight loss assessment,and the time of thehistology evaluations.(B) Kaplan-Meier plotof the percent survivalas a function of timefrom cisplatin adminis-tration. Data are fromn = 8 for all groups ex-cept for PBS (n = 5) andfCNT/siCtr1 (n = 7). Pvalues are in table S4.(C) Forest plot of thehazard ratios of the var-ious prophylactic groupsversus the combinationfCNT/siTrp53/siMep1b.(D) Comparison of theH&E staining at day 11of the kidney cortex ofa representative mousereceiving fCNT/siTrp53/siMep1b and cisplatin-induced nephrotoxicinsult versus a naïvecontrol mouse. Scalebars, 50 mm. (E) Quan-tification of picrosiriusred staining for kidneyfibrosis at day 11 or180 after cisplatin ad-ministration and repre-sentative images. Dataare means ± SEM (n =10 at day 11; n = 12 forfCNT/siScram and n = 8for fCNT/siTrp53/siMep1bat day 180). Scale bars,200 mm. (F) CD3 immu-nostaining for T cellinfiltration and repre-sentative images. Dataare means ± SEM (n =9 for all groups exceptfor fCNT/siTrp53/siMep1b at day 180 when n = 6). (G) Analysis of the lymphocyte infiltration by CD45 staining. Data are means ± SEM (n = 9 for all groups). (H) Analysis ofmacrophage infiltration by Iba1 staining. Data are means ± SEM (n = 9 for all groups). For (F) to (H), P values were determined by unpaired t test.Scale bars, 20 mm.

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is no U.S. Food and Drug Administration–approved pharmaceuticalfor the prevention or treatment of AKI, which is associated to veryhigh rates of mortality and morbidity (20). Other platforms forRNA delivery often involve complex aggregate structures, syntheticRNAi modification, intricate formulations, and multiple purificationsteps. The fCNT platform is a single molecule that can be loadedwith RNA in a predetermined stoichiometry with nanomolar bind-ing affinity (13). Assembly is rapid (<1 min) and no separations arenecessary. Looking forward to clinical use, this drug could also beassembled with a tracer-labeled siRNA component to PET imageand assess biodistribution and clearance to determine RNAi dose de-livered to tissue.

fCNT is an ideal delivery vehicle for siRNA because the uniquemolecular platform protects noncovalently loaded siRNA from serumdegradation. It is even more ideal for RNAi in kidney-related diseasesbecause the soluble fCNT naturally traffics to the kidney, with rapidclearance in vivo (9, 10). These nanotubes rapidly cleared from theblood (t1/2 = 6 min) and were readily filtered by the glomerulus. Thus,for the first time, a macromolecular-sized nanomaterial platform canbe used to specifically deliver effective doses of siRNA to the kidney,whereas other nanomaterials are hepatically sequestered. PK profil-ing of fCNT-bearing radiotracer-labeled siRNA showed that 10-foldmore siRNA cargo was distributed to the kidney compared to thesiRNA alone.

The siRNA-laden construct remained intact, and the fCNT di-rected clearance and distribution of the cargo. Cell binding data dem-onstrated that the siRNA cargo was internalized and ultimately releasedin accordance with our proposed off-loading mechanism (13). Con-focal imaging data also described PTC internalization and traffickingof fCNT in vivo that was consistent with a clathrin-mediated endocyticuptake after capture at the brush border (28–30). PET/CT imaginganalyses of 86Y tracer–labeled fCNT in nonhuman primates showed aPK profile that was nearly identical to the distribution and clearance inmice, suggesting that this molecular platform could be translated forhuman use. Other than targeting the kidney, the fCNT delivery vehi-cle improved the RNAi process compared with free siRNA becausefCNT/siRNA was able to knock down 75% of EGFP as well as Ctr1, acopper-ion transporter in vivo.

Epithelial cells lose their polarity and apoptose after nephrotoxicinsult (15–19). PTCs are known to accumulate siRNA, and severalstudies have proposed RNAi as a strategy to counteract AKI patho-genesis (19), highlighting the potential of systemically administeredsiRNA for the treatment of kidney disorders in clinical practice (35, 36).Stabilized siRNA that inhibits P53 expression is in early developmentfor AKI therapy (ClinicalTrials.gov: NCT00554359 and NCT00802347)and is one of the first systemically administered siRNA drugs to enterclinical trials. Furthermore, the involvement of meprin in kidney in-jury has been shown in knockout mice and through meprin inhibi-tors, both providing protection against AKI and improving renalfunction (21). Our strategy was to deliver siRNA to the kidney to re-duce the expression of two proteins with demonstrated roles in AKI,meprin-1b and p53 (17–19, 21–26).

Our data confirmed the ability of fCNT to effectively deliver siMep1band siTrp53 to PTCs and significantly reduce the expression of thesetwo target genes and their respective proteins. These experiments werealso designed to assess safety and showed that the fCNT and siRNAwere biocompatible with no observed renal (or other) toxicity. ThefCNT/siTrp53/siMep1b combination was able to maintain the message

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and protein expression for p53 and meprin-1b at baseline levels after anephrotoxic insult and pharmacologically spare mice from renal injury.We identified a pharmacological intervention for a dose of nephrotoxiccisplatin that improved progression-free survival, reduced fibrosis, anddecreased immune cell infiltration more than control treatments. Kid-neys from animals treated with fCNT/siTrp53/siMep1b showed signif-icantly lower levels of macrophage, leukocyte, and T cell infiltrationwithin the kidney cortex at 11 and 180 days after cisplatin injury, whichindicates not only immediate disease resolution but also a durable effect.Combination fCNT-mediated RNAi also reduced chronic fibrosis.

The mechanism of drug action required the simultaneous targetingand down-regulation of siMep1b and Trp53 expression in the renalproximal tubule cells to significantly reduce kidney injury and prolongsurvival. The ability of fCNT-mediated delivery of a combination ofsiMep1b and siTrp53 to protect mice from renal injury resulted fromtargeted mRNA degradation of two genes that contribute to loss of epi-thelial cell polarity and apoptosis; the up-regulation of either can initiateinjury. Individually, each siRNA (with or without fCNT) was insuffi-cient to limit injury at the cumulative 0.4 mg siRNA/kg doses that weused in this work. These findings suggest that the loss of polarity andapoptosis in PTC were distinct co-events that can each contribute toinjury, but the coadministration of siMep1b and siTrp53 provided asynergistic therapeutic effect.

Therapeutic single gene (P53) interference was reported to mitigatecisplatin injury in rat kidneys (19). However, in our study, siTrp53(delivered with or without fCNT) was unable to control injury, whereasthe combination with siMep1b delivered by the fCNT was effective. Weattribute the differences in these two studies to the following: siRNAdoses differing 90-fold, with cumulative dose of 36 mg/kg in (19) and0.4 mg/kg in our study; our use of natural RNA versus stabilized, syn-thetic RNA in (19); animal model [8-week-old rats in (19) versus 49-to 52-week-old mice in our study]; and degree of injury, where we used10 mg/kg cisplatin versus 7.5 mg/kg in (19).

Because drug-induced nephrotoxicity is responsible for nearlyone-fifth of AKI cases (37), we investigated the use of dual RNAi ina rodent model of cisplatin injury in a prophylactic treatment setting.Although cisplatin is often used in the context of cancer, one limita-tion of our study was the use of healthy but elderly animals. Althoughit is possible that tumors could alter the PKs of the fCNT platform(that is, by nonspecific accumulation in tumor), we do not expect thisto be a major impact owing to the lack of a targeting moiety on thefCNT/siRNA (11, 12, 28). We also investigated only one model of renalinjury, others such as I/R injury should be studied to assess the broadimpact of this siRNA therapeutic approach.

Nano-mediated RNAi as a treatment for AKI seeks to transformthe way in which a nephrotoxic renal insult could be managed clini-cally to avoid injury. Our fibrillar nanocarbon agent with dual siRNAsignificantly improved progression-free survival in a clinically relevantmodel of cisplatin-induced AKI and was safe in nonhuman primates.fCNT therefore represents a nanomedicine tool to enable robust pro-phylactic therapy to mitigate and minimize AKI with widespreadapplication to patients receiving pharmaceutical regimens with neph-rotoxic side effects. Such an approach may also afford the capacity toincrease the therapeutic index while protecting the kidney (which iscommonly a dose-limiting organ). Moreover, this technology can serveas a precision tool in the study of biological pathways in the nephronand aid in selecting appropriate targets to facilitate the drug designprocess. The translation of this work into a preventive therapeutic

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opportunity for patients will require the cGMP (current GoodManufacturingPractices) production of fCNT, a comprehensive cGMP toxicity eval-uation of the fCNT/siRNA drug in rodents and larger animal models,the investigation of different models of AKI, and clinical trials to assesssafety, compatibility, and PKs in patients at high risk of nephrotoxicinjury.

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MATERIALS AND METHODS

Study designThe purpose of this study was to investigate the therapeutic effect offibrillar nanocarbon (fCNT)–mediated RNAi in a rodent model of neph-rotoxic AKI by targeting two genes that are overexpressed in humansin response to renal injury, P53 and MEP1B. The use of fCNT wasexpected to selectively target the proximal tubule cells and reducethe siRNA dose, avoid off-target effects, and protect siRNA drug car-go. This model could be translated into humans as a preventive ther-apy in anticipation of a nephrotoxic insult. Age-related morbidity wasintroduced by using 49- to 52-week-old Balb/c mice. Mice were ran-domized and treated first with the fCNT/RNAi drug or controls 3 daysbefore renal injury was induced by administering cisplatin intra-peritoneally. Blood was collected on days 1, 5, 8, and 11 (after cisplatinadministration) to assay biomarkers of kidney injury; weight changeswere recorded daily; and observations of lethargy or death were noted.Progression-free survival was analyzed using the Kaplan-Meier methodto score outcomes of weight loss (≥20% of initial mass), renal bio-marker values (≥3 SDs relative to untreated group mean), severe leth-argy, or death. Three representative mice from each group weresacrificed on day 11 (endpoint for acute injury in survival experiments)and the remainder on day 180 (chronic injury time point). Kidneyswere harvested, fixed, and embedded in paraffin for histological analysisof fibrosis, T cell, lymphocyte, and macrophage infiltration. Weights,serum samples, and ROIs for IHC and IF images were recorded andanalyzed blindly. Sample size was determined on the basis of our pre-liminary data (fig. S9) and on previous studies (19, 38, 39). After setendpoints for the survival experiment were reached, the weights increasedand blood chemical parameters returned to normal for all the survivinganimals. No outliers were selected in any of the experiments.

CNT generation and characterizationHigh-pressure carbon monoxide–produced SWCNTs were processedto yield SWCNT-NH2 as previously described (9–13). Purity and iden-tity of the fCNT were assessed by ultraviolet-visible spectroscopy, HPLC,TEM, and DLS (dynamic light scattering) as described (9, 10). Com-plete details are reported in the Supplementary Materials.

siRNA sequencesDicer-validated RNA sequences designed (27) to target enhanced greenfluorescent protein (EGFP), murine copper transport protein 1 (Ctr1),meprin-1b (Mep1b), and p53 (Trp53) were obtained from IntegratedDNA Technologies Inc. along with a nonspecific scrambled sequence(Scram) and are reported in the Supplementary Materials.

AnimalsAll animal studies were approved by the Institutional Animal Care andUse Committee of Memorial Sloan Kettering Cancer Center (MSKCC).The experiments used female Balb/c mice (Taconic) aged 6 to 7 weeks

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or 49 to 52 weeks; male nu/nu aged 8 to 12 weeks (Taconic); and femaleC57BL/6 Trp53 null and female C57BL/6 wild type (Jackson Labs) 6 to8 weeks old. The b-actin–EGFP transgenic C57BL/6 mice were providedby the Joyce laboratory at MSKCC (female, 8 to 10 weeks old). Malecynomolgus monkeys (Charles River Laboratories) were 3 years old.

Cell cultureHeLa cells expressing EGFP (EGFP+ HeLa, Cell Biolabs) were culturedat 37°C and 5% CO2 in high-glucose Dulbecco’s modified Eagle’s me-dium (Life Technologies) supplemented with 10% fetal bovine serum(Life Technologies), 0.1 mM MEM nonessential amino acid solution(Life Technologies), 2 mM L-glutamine (Life Technologies), andblasticidin (0.010 mg/ml) (Life Technologies).

Kinetics of EGFP+ HeLa cell internalization of fCNT/siRNAInternalization kinetics of fCNT/siEGFP was evaluated and quantifiedin EGFP+ HeLa cells. Confocal microscopy was used to image inter-nalization in real time and utilized the fluorescent siEGFP-Cy3sequence; radionuclide-based internalization of siEGFP-[111In]DOTAwas quantified using a cell-stripping assay.

Proximal tubule cell trafficking experimentsMice received fCNT-(AF488)(AF680)(DOTA) or controls which in-cluded vehicle, only the AF488 dye. Harvested tissue was fixed over-night in 4% PFA at 4°C, embedded in paraffin, and sectioned to obtain0.005-mm-thick samples. IF staining details are described below andin table S5. Widefield microscopy was performed with an Axioplan2imaging microscope equipped with AxioCam MRm camera (Zeiss),using filter cubes for DAPI, AF488, and TRITC (tetramethylrhodamineisothiocyanate). Confocal microscopy was performed using an InvertedLeica TCS SP5 microscope (Leica). All three-dimensional (3D) render-ing has been done with Imaris (Bitplane).

fCNT-mediated RNAi in vitroEGFP+ HeLa cells were used to investigate fCNT/siEGFP silencingin vitro using flow cytometry, confocal microscopy, Western blotanalyses, and quantitative RT-PCR (qRT-PCR) versus controls. Flowcytometry was used to investigate the change in green fluorescenceintensity in cells. Time-lapse microscopy was used to image the changein green cell fluorescence in real time. Western blot analysis was usedto measure EGFP protein expression. qRT-PCR analysis was used tomeasure EGFP expression.

In vitro cytotoxicity experimentEGFP+ HeLa cells were treated with fCNT or controls, and viabilitywas evaluated by flow cytometry using propidium iodide (Life Tech-nologies) to detect dead cells.

Immunohistochemical and immunofluorescence stainingKidney tissue sections were stained using a Discovery XT processor(Ventana Medical Systems) in the MSKCC Molecular Cytology CoreFacility. Reagents and protocol details are listed in table S5.

PK studies of fCNT/siEGFP-[111In]DOTAand [86Y]fCNTBiodistribution studies of fCNT/siEGFP-[111In]DOTA versus siEGFP-[111In]DOTA alone examined the heart, kidneys, lung, spleen, liver,stomach, intestine, muscle, bone, blood, and urine. Standards of the

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injected formulation were counted to determine the %ID and %ID pergram of tissue. Samples of the injected formulations and urine samplesfrom each group were analyzed by HPLC. Dynamic PET/CT was per-formed with a Siemens Biograph mCT PET/CT system (40). Images inFig. 3 (D and E) were elaborated as 3D volume rendering of PET dataoverlaid onto surface-rendered CT data. [86Y]fCNT was prepared andcharacterized as previously described (31).

fCNT persistence in the kidneys as a function of timeAnimals (4- to 6-week-old female Balb/c mice) received an intravenousdose of 0.1 ml of fCNT-AF488 (0.25 g/liter in PBS). Mice were sacri-ficed at 1, 3, 7, or 30 days after injection, and kidneys were fixed in PFAand paraffin-embedded for IF staining.

Dose escalation and renal accumulation offCNT/siEGFP-[111In]DOTAThe kidney accumulation of fCNT/siEGFP-[111In]DOTA was investi-gated as a function of dose and schedule.

EGFP knockdown in vivoThis experiment used b-actin–EGFP transgenic C57BL/6 mice that re-ceived fCNT/siEGFP or controls. Each animal per group received a daily0.22-ml intravenous injection of the respective drug or control for threeconsecutive days. Mice were sacrificed 1 day after the last injection. Tis-sues were harvested and fixed-frozen for histological studies. Imageswereacquiredwith an inverted fluorescencemicroscope (NikonTi Eclipse runwith NIS-Elements Ar) and processed with Fiji (41). ROI analysis wasdoneon×20magnificationof 0.010-mm-thick sections imagedwithwhitelight (DIC-like), DAPI, and GFP channel. About 50 tubules per experi-mental or control imagewere quantified (more than 300 cells per group).

Ctr1 knockdown and copper-64 uptake into kidneys in vivoMice received fCNT/siCtr1 or controls every day for three consecutivedays. On the third day, every animal received an intravenous injectionof 133 kBq of 64CuCl2 (Washington University) and were sacrificed1 hour later. The kidneys, liver, heart, and blood were harvestedand weighed, and radioactivity was measured on a g-counter. The %ID/g was evaluated by comparison with known standards.

fCNT-mediated knockdown of p53 and meprin-1b in vivoFemale Balb/c mice were grouped as follows: (i) fCNT/siMep1b (n = 7);(ii) fCNT/siTrp53 (n = 7); (iii) siMep1b (n = 7); (iv) siTrp53 (n = 7); and(v) PBS vehicle (n = 3). Each animal received a daily 0.10-ml intravenousinjection of 0.032 mg of the 1:1 (mol/mol) fCNT/siRNA constructs,0.002 mg of the siRNA alone, or the PBS vehicle for three consecutivedays. Renal health was assessed on day 4 using a metabolic panel thatassayed blood urea nitrogen, serum creatinine, phosphorus, and mag-nesium. Kidneys were harvested on day 4, fixed, sectioned, and stainedwith H&E to examine tissue morphology as a function of treatment.Tissue morphology was examined and scored blindly by an institution-al veterinary pathologist. The expression of meprin-1b and p53 in therenal cortex was evaluated using IHC and quantitative ROI analysis.

mRNA and protein expression levels in mice that receivedfCNT/RNAi prophylaxis and cisplatinMice were treated for five consecutive days with a daily dose of fCNT/siTrp53/siMep1b (1.6-mg fCNT + 0.087-mg siRNA per kilogram),fCNT/siScram, or PBS. Cisplatin (10 mg/kg dose) was intraperito-

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neally administered at day 3 to the fCNT/siTrp53/siMep1b and fCNT/siScram groups. Animals were sacrificed on day 6; kidneys were har-vested, and half of each kidney was sectioned and fixed in PFA forIHC and FISH (Affymetrix) analyses. The remaining half washomogenized in a TissueLyser II (Qiagen) with radioimmunoprecipita-tion assay buffer and protease inhibitor to extract protein. Trp53 andMep1b expression levels were measured in kidney cortex homogenatesusing a pan p53 ELISA kit (Roche Applied Science) and pan meprin-1b ELISA kit (MyBioSource), respectively, according to the manufac-turer’s instructions. mRNA was extracted and purified with the RNeasyPlus Mini kit (Qiagen) according to the manufacturer’s protocol. Ex-pression of the Trp53 and Mep1b was assayed by qRT-PCR.

Progression-free survival after fCNT/RNAi prophylaxisProgression-free survival was evaluated in mice prophylactically treatedto silence the renal expression of Trp53,Mep1b, and Ctr1. Each animalreceived a daily dose of 1.6-mg fCNT + 0.087-mg siRNA per kilogram(1:1 mol/mol) or 0.087 mg siRNA/kg or PBS vehicle in 0.10 ml by in-travenous injection for 5 days. Cisplatin (Sigma; 10 mg/kg in normalsaline solution) was administered on day 3. Here, nine groups of femaleBalb/c mice were arranged as follows: (i) PBS vehicle (n = 5); (ii) fCNT/siMep1b (n = 8); (iii) fCNT/siTrp53 (n = 8); (iv) fCNT/siScram (n = 8);(v) siMep1b (n = 8); (vi) siTrp53 (n = 8); (vii) a combination of fCNT/siTrp53/siMep1b (n = 8); (viii) a combination of siTrp53/siMep1b (n = 8);and (ix) fCNT/siCtr1 (n = 7). Parameters and analysis of the progression-free survival are described in Study design.

Statistical analysisGraphPad Prism 6 was used for all statistical analyses. Student’s un-paired t test was used for statistical comparisons between groups. Log-rank (Mantel-Cox) test was used for the survival curve comparison, and95% confidence intervals from the Mantel-Haenszel test were used forthe hazard ratios. P values of <0.05 were considered significant.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/331/331ra39/DC1MethodsFig. S1. Amine content and size distribution of fCNT.Fig. S2. fCNT-mediated siRNA cellular internalization in vitro.Fig. S3. Fluorescence intensity and mRNA expression over time during fCNT-mediatedknockdown of EGFP in vitro.Fig. S4. In vitro dose-dependent cytotoxicity study.Fig. S5. fCNT delivers siRNA to the kidneys in mice.Fig. S6. In vivo stability study of the fCNT/siRNA complex.Fig. S7. Kidney morphology after treatment with just the prophylactic agents in the absence of cisplatin.Fig. S8. Trp53 and Mep1bmRNA expression in cortical PTC during cisplatin-induced nephrotoxic insult.Fig. S9. Cisplatin dose response.Fig. S10. fCNT/siRNA dose escalation study.Table S1. Blood chemistry during prophylactic treatment.Table S2. Weight values for the progression-free survival study.Table S3. Blood chemistry values for the progression-free survival study.Table S4. Progression-free survival data from the Kaplan-Meier analysis.Table S5. Staining conditions.

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Acknowledgments: We thank R. Bowman and J. Joyce for the EGFP mice; M. Fleisher for ad-vice on the renal biomarker studies; E. Skolnik and E. Jaimes for expert opinion on renal bi-ology and pathology; J. Lewis for the copper-64; J. Gardner for help with the survival studies;A. C. McDevitt for the graphic illustration; M. Kharas for the critical reading of the manuscript;and M. Turkekul, N. Fan, D. Yarilin, A. Barlas, and M. Brendel from the Molecular Cytology CoreFacility. Funding: This work was supported by the Office of Science [BER (Biological andEnvironmental Research)], U.S. Department of Energy (award DE-SC0002456), NIH MSTP (Med-ical Scientist Training Program) (grants GM07739, R21CA128406, R01CA166078, R01CA55349,R25TCA046945, R24CA83084, P30CA08748, P01CA33049, and F31CA167863), Memorial SloanKettering Center for Molecular Imaging and Nanotechnology (CMINT), and Memorial SloanKettering Experimental Therapeutics Center. Author contributions: M.R.M. and S.A. conceivedthe study; designed and executed the functionalization and characterization of fCNT, fCNT/siRNA binding studies, radiolabeling, and in vivo experiments; analyzed data; and wrote thepaper. N.A. performed in vitro experiments, RT-PCR, Western blotting, and flow cytometryanalysis, and contributed to the in vivo EGFP knockdown study, the 64Cu study, the biodistri-bution, the cisplatin dose-response experiment, the cisplatin response experiment, and thesurvival experiment. D.L.J.T. contributed to the writing and provided Figs. 2A and 3D. K.B.assisted in the nonhuman primate imaging experiment. Y.R. and K.M.-T. contributed to theacquisition and analysis of IHC and confocal images. D.Q. performed RT-PCR experiments. B.J.B.provided settings for the PET/CT imaging and helped analyze the data. M.B. performed DLSand TEM analysis. D.A.S. discussed experimental design, analyzed the data, and helped writethe manuscript. Competing interests: A patent was filed in July 2015: “Method andcomposition for targeted delivery of therapeutic agents.” Data and materials availability:All data and materials are available.

Submitted 6 July 2015Accepted 29 February 2016Published 23 March 201610.1126/scitranslmed.aac9647

Citation: S. Alidori, N. Akhavein, D. L. J. Thorek, K. Behling, Y. Romin, D. Queen, B. J. Beattie,K. Manova-Todorova, M. Bergkvist, D. A. Scheinberg, M. R. McDevitt, Targeted fibrillarnanocarbon RNAi treatment of acute kidney injury. Sci. Transl. Med. 8, 331ra39 (2016).

eTranslationalMedicine.org 23 March 2016 Vol 8 Issue 331 331ra39 11

Targeted fibrillar nanocarbon RNAi treatment of acute kidney injury

Katia Manova-Todorova, Magnus Bergkvist, David A. Scheinberg and Michael R. McDevittSimone Alidori, Nima Akhavein, Daniel L. J. Thorek, Katja Behling, Yevgeniy Romin, Dawn Queen, Bradley J. Beattie,

DOI: 10.1126/scitranslmed.aac9647, 331ra39331ra39.8Sci Transl Med

monkeys. The next steps will be testing the dual siRNAs in other animal models of kidney injury.given both siRNAs. The nanotube/siRNA complexes were also safe and had favorable pharmacokinetics in

two key proteins involved in kidney injury; the mice lived longer and remained injury-free, but only if−−and p53βmice before drug-induced kidney insult. With such RNA interference, the kidney cells could not produce meprin-1

were attached to fibrillar carbon nanotubes and delivered simultaneously toTrp53 and Mep1bsiRNAs targeting delivers two small interfering RNAs (siRNAs) to the main cells of the kidney, the renal proximal tubule cells.

. devised a nanomedicine treatment approach thatet alrobs the organs of oxygen. To prevent injury, Alidori The kidneys can be damaged by drugs, such as antibiotics and chemotherapy, as well as by surgery, which

Double trouble for kidney toxicity

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